Identification and Characterization of a Novel Protein–Protein Interaction Among SARS‐CoV‐2 Nucleocapsid, Host SFPQ, and hnRNP U and Its Potential Role in Virus Replication

SARS-CoV-2 has led to significant global health and economic challenges and caused the COVID-19 pandemic. The ability of the virus to replicate adeptly within host cells is critical for its pathogenicity. The structural nucleocapsid (N) protein of SARS-CoV-2 packages newly synthesized viral RNA with the association of various host proteins that may contribute to different functions in maintaining a productive viral life cycle. In this study, we report the identification and characterization of host proteins SFPQ and hnRNP U interacting with SARS-CoV-2 N protein in both N-transfected cells and virus-infected cells, forming a hetero-trimeric protein complex. Using carefully designed peptides that span the length of N protein and competitive inhibition, we identified the interacting domains at N protein that interact with SFPQ and hnRNP U. Our results constitute the first report that the characterized N protein and host SFPQ and hnRNP U form a hetero-trimeric protein complex in both N transfected cells and virus-infected cells. Utilizing competitive peptides, we were able to disrupt the hetero-trimeric protein complex in virus-infected cells, leading to reduction in viral replication. These results clearly demonstrate that N-SFPQ-hnRNP U hetero-trimeric protein complex formation is found in SARS-CoV-2 infected cells that regulate viral replication. Our findings suggest that the protein-protein interaction (PPI) between N-SFPQ-hnRNP U hetero-trimeric protein complexes could be a novel drug target for developing therapeutics against COVID-19.